An mRNA complex vaccine with yeast cell wall particles as adjuvant and application thereof

Abstract

The present application provides a kind of mRNA complex vaccine with yeast cell wall particles as adjuvant, belongs to the technical field of immunotherapy, and the complex vaccine includes yeast cell wall particles, cationic lipid nanoparticle loaded with tumor antigen coding mRNA, or includes PEI modified yeast cell wall particles, lipid nanoparticle loaded with tumor antigen coding mRNA;The complex vaccine of the present application acts in TC-1 tumor-bearing mouse model, and the complex vaccine can significantly inhibit tumor growth;IFN-γ+CD8+CD3+Cell ratio and IFN-γ ELISPOT spot forming cell number in the complex vaccine group are significantly increased;Among them, the complex vaccine PEI-YCWP+LNP-HPV E7 can effectively induce the maturation and cross presentation of dendritic cells, activate antigen-specific CD8+T cell response, and enhance NK cell function by inducing training immunity, to realize the synergistic activation of innate immunity and adaptive immunity.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to an mRNA complex vaccine using yeast cell wall particles as adjuvant and its application in the treatment of tumors. Background Technology

[0002] mRNA vaccines have shown great potential in infectious disease prevention and tumor immunotherapy due to their advantages such as flexible design, rapid preparation, and ability to encode any antigen. Lipid nanoparticles (LNPs) are currently the most mainstream mRNA delivery vector and have been successfully validated in COVID-19 mRNA vaccines from companies such as Moderna and BioNTech. However, the immunogenicity of mRNA vaccines still faces many challenges: naked mRNA is easily degraded, antigen presentation efficiency is limited, and it is difficult to effectively activate innate immune responses.

[0003] Adjuvants are key factors determining the strength and type of vaccine-induced immune responses. In recent years, trained immunity has received widespread attention as a novel immune regulation strategy. Trained immunity refers to an "immune memory" state acquired by innate immune cells after initial stimulation through epigenetic and metabolic reprogramming, enhancing their ability to respond to subsequent stimuli. β-glucan has been proven to serve as an inducer of trained immunity and a vaccine adjuvant.

[0004] Yeast cell wall particles (YCWPs) are derived from the cell walls of *Saccharomyces cerevisiae*, and their main component is β-1,3-D-glucan. GPs are hollow, porous microparticles extracted from yeast cell walls. Due to their unique hollow structure and inherent immunomodulatory properties, they have been developed as a drug delivery platform. Currently, YCWPs are used to load protein antigens and deliver them to macrophages and dendritic cells, and are also used for in-situ synthesis of DNA nanoparticles within the lumen for gene delivery. However, none of these applications involve mRNA delivery, and their technological pathways cannot be directly transferred to the field of mRNA delivery. mRNA molecules possess physicochemical properties drastically different from protein antigens and DNA—their single-stranded flexible conformation, extremely high negative charge density (one negative charge per nucleotide), and chemical instability due to their susceptibility to degradation by ribonucleases pose entirely different requirements for charge matching, structural protection, and endosome escape efficiency in mRNA delivery. In particular, natural YCWP carries a negative charge on its surface, and mRNA also carries a negative charge. There is electrostatic repulsion between the two, which makes it impossible for natural YCWP to effectively load mRNA, severely limiting its application as an adjuvant / delivery vector for mRNA vaccines.

[0005] Polyethyleneimine (PEI) is a cationic polymer that has been used in the synthesis of DNA nanoparticles within yeast cell wall particles. There are no existing reports of using PEI-modified YCWP as an adjuvant in the preparation of vaccines with mRNA-LNP antigens, nor is there any explanation of its mechanism for enhancing the immune response of mRNA vaccines through immunization training. Summary of the Invention

[0006] This invention provides an mRNA complex vaccine using yeast cell wall particles as adjuvant. This invention solves the charge mismatch problem between yeast cell wall particles (YCWP) and lipid nanoparticles (LNP) by preparing cationic lipid nanoparticles loaded with tumor antigen mRNA, or by modifying yeast cell wall particles with PEI, thereby achieving efficient complexation of adjuvant and antigen.

[0007] The present invention provides an mRNA complex vaccine using yeast cell wall particles as adjuvant, comprising yeast cell wall particles (YCWP) and cationic lipid nanoparticles (DLNP-mRNA) loaded with mRNA encoding tumor antigens, wherein the mass-to-volume ratio of YCWP to DLNP-mRNA is 1:1-2 mg:mL.

[0008] The yeast cell wall particles (YCWP) were prepared using conventional methods, for example, according to the method described in the literature “Hunter, KW, Jr., Gault, RA, & Berner, MD (2002). Preparation of microparticulate beta-glucan from Saccharomyces cerevisiae for use in immune potentiation. Letters in Applied Microbiology, 35(4), 267-271.”

[0009] The cationic lipid nanoparticles loaded with mRNA encoding tumor antigens were prepared by dissolving (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), distearate phosphatidylcholine (DSPC), cholesterol, methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159), and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in anhydrous ethanol using a microfluidic device. The lipid mixture was mixed with citrate buffer encoding tumor antigen mRNA, and the mixture was dialyzed and filtered through a 0.22 μm filter for sterilization to obtain the following molar ratios: DOTAP to DSPC 10⁻¹²:30⁻³⁵; DOTAP to cholesterol 10⁻¹²:9⁻¹⁰; DOTAP to ALC-0159 10⁻¹²:40⁻⁴⁵; and DOTAP to ALC-0315 10⁻¹²:1⁻².

[0010] The mRNA complex vaccine of the present invention, which uses yeast cell wall particles as adjuvants, may further include PEI-modified yeast cell wall particles (PEI-YCWP) and lipid nanoparticles (LNP-mRNA) loaded with mRNA encoding tumor antigens; the mass-to-volume ratio of PEI-YCWP to LNP-mRNA is 1:1-2 mg:mL.

[0011] The molecular weight of PEI (polyethyleneimine) in PEI-modified yeast cell wall particles is 25,000 Da.

[0012] In the above-mentioned composite vaccine, the adjuvant component and the antigen component are bound together by electrostatic adsorption to form a composite vaccine; YCWP binds to DLNP-mRNA by electrostatic adsorption, and PEI-YCWP binds to LNP-mRNA by electrostatic adsorption to form PEI-YCWP+LNP-mRNA or YCWP+DLNP-mRNA complex.

[0013] The PEI-modified yeast cell wall particles were prepared using conventional methods, such as those described in the literature “Yang, F. etal. (2021). Polyethyleneimine-complexed charge-reversed yeast cell walls for the enhanced oral delivery of pseudovirus-based antigens. Chemical Communications, 57(95), 12768-12771.”

[0014] The lipid nanoparticles loaded with tumor antigen mRNA were prepared using conventional methods, such as those described in the literature “Nature Protocols (2025): mRNA lipid nanoparticle formulation, characterization and evaluation”.

[0015] Another objective of this invention is to apply the above-mentioned mRNA complex vaccine with yeast cell wall particles as adjuvant in the preparation of antitumor drugs.

[0016] The mRNA encoding the tumor antigen is the mRNA encoding the HPV E7 antigen, and its nucleotide sequence is shown in SEQ ID NO:1. The tumor is cervical cancer.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention confirms that yeast cell wall granule adjuvant can enhance antigen expression in RAW264.7 macrophages, and constructs a composite vaccine platform that combines training immune induction and antigen delivery functions; This invention modifies the YCWP surface to carry a positive charge (the Zeta potential flips from about -9.5mV to +29mV), which solves the technical problem that natural YCWP cannot effectively load negatively charged LNP-mRNA. Electron microscopy results confirm that a large number of nanoscale LNP particles are attached to the surface of PEI-YCWP. In RAW264.7 macrophages, the expression level of luciferase in the PEI-YCWP+LNP-mRNA group was approximately double that in the LNP-mRNA group, and the expression level in the YCWP+DLNP-mRNA group was also approximately double that in the DLNP-mRNA group. This result is the first to demonstrate that yeast cell wall granules or PEI-modified YCWP, as adjuvants, can enhance the uptake or endosome escape efficiency of macrophages of LNP-mRNA (DLNP-mRNA), suggesting that YCWP enhances cellular uptake or endosome escape and improves translation efficiency. YCWP adjuvants possess antigen-presenting cell-targeting properties and can be extended to antigen systems. 2. This invention reveals that PEI-modified YCWP and LNP-mRNA are saturated and activated, with no additional contribution from the antigen, proving that the synergy is not a global signal enhancement but a pathway-selective process; 3. This invention validated in vitro the enhancement of LNP-mRNA response induced by PEI-modified YCWP-induced training immunity, and further confirmed in vivo the establishment of systemic training immunity. In vitro experiments showed that after 7 days of rest, BMDCs trained with the combined vaccine showed a CD40+ ratio of 49.8% in response to LNP-mRNA challenge (22.5% in the PBS training group, p<0.0001) and IL-12p70 secretion of 67.6 pg / mL (2.4 times that in the PBS training group, p<0.01); TNF-α and IL-6 secretion in response to LPS challenge increased by approximately 2.5 times (p<0.0001), demonstrating that YCWP adjuvant-induced training immunity has antigen specificity in the form of mRNA. In vivo experiments further showed that the uptake of 2-NBDG in the bone marrow MPP of mice in the combined vaccine group was significantly enhanced (p<0.01), proving that the PEI-modified YCWP adjuvant established a systemic training immunity at the hematopoietic stem cell level. In a TC-1 tumor-bearing mouse model (using HPV E7 as the model antigen), the combined vaccine of this invention showed that the tumor volume in the combined vaccine group was significantly smaller than that in the PBS group from day 15 (p<0.01), and the tumors of some mice completely regressed (3 / 8 mice). Immunological analysis showed that the maturation of dendritic cells (DCs) and the IFN-γ production capacity of NK cells in the combined lymph nodes were enhanced. 4. This invention provides a new strategy for the development of mRNA vaccine adjuvants, which has significant clinical application prospects and industrialization value; This invention links yeast cell wall particles to an mRNA vaccine, achieving a synergistic effect of "1+1>2" immune enhancement through the combined action of training immunity induced by PEI-modified YCWP adjuvant and adaptive immunity induced by LNP mRNA antigen. This invention provides a novel solution to overcome the bottleneck of insufficient immunogenicity in existing mRNA vaccines, and has broad clinical application prospects and industrialization value. Attached Figure Description

[0018] Figure 1 The results of zeta potential detection for YCWP, PEI-YCWP, YCWP+DLNP-HPV E7 and PEI-YCWP+LNP-HPV E7 complexes; Figure 2 The results of zeta potential detection for LNP-HPV E7 and DLNP-HPV E7; Figure 3 Transmission electron microscopy images of YCWP (A), LNP-HPV E7 (B), and DLNP-HPV E7 (C); Figure 4 Transmission electron microscopy images of the PEI-YCWP+LNP-HPV E7 complex (A) and the YCWP+DLNP-HPV E7 complex (B); Figure 5Results of luciferase mRNA transfection efficiency assays in RAW264.7 cells using different formulations; Figure 6 Tumor growth curves (A) and tumor images (B) for each group in the TC-1 tumor-bearing mouse model. Figure 7 The statistical results show the number of IFN-γ+ spots in tumor tissue; Figure 8 The results of flow cytometry analysis of the proportion of CD86+ in CD11c+ in lymph nodes; Figure 9 The results are from flow cytometry analysis of the ratio of IFN-γ+CD8+CD3+ in lymph nodes. Figure 10 The results of flow cytometry analysis of the proportion of IFN-γ+ in CD3-NK1.1+ in lymph nodes; Figure 11 The results of flow cytometry analysis of the proportion of MPP cells in LSK cells in bone marrow HSPCs; Figure 12 Flow cytometry results of 2-NBDG uptake in bone marrow MPP; Figure 13 The results of flow cytometry analysis of p-IRF3 phosphorylation levels at different time points after BMDC was stimulated by different formulations; Figure 14 The results of flow cytometry analysis of p-Syk phosphorylation levels at different time points after BMDC was stimulated by different formulations; Figure 15 ELISA results of TNF-α secretion after LPS / PBS challenge following BMDC training immunization; Figure 16 ELISA results of IL-6 secretion after LPS / PBS challenge following BMDC training immunization; Figure 17 ELISA results of IL-12p70 secretion after LPS / PBS challenge following BMDC training immunization; Figure 18 Flow cytometry results of the CD11c+ CD40+ ratio after LPS / PBS challenge following BMDC training immunization; Figure 19 Flow cytometry results of the CD11c+ CD86+ ratio after LPS / PBS challenge following BMDC training immunization; Figure 20 Flow cytometry results of CD11c+ MHC-II+ ratio after LPS / PBS challenge following BMDC training immunization; Figure 21ELISA results of IL-12p70 secretion after LNP-HPV E7 / PBS challenge following BMDC training immunization; Figure 22 ELISA results of IL-6 secretion after LNP-HPV E7 / PBS challenge following BMDC training immunization; Figure 23 Flow cytometry results of the CD11c+ CD40+ ratio in LNP-HPV E7 / PBS challenge after training immunization for BMDC; Figure 24 Flow cytometry results of the CD11c+ CD86+ ratio after LNP-HPV E7 / PBS challenge following BMDC training immunization. Detailed Implementation

[0019] The present invention will be further described below through specific embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the methods in these embodiments are conventional methods, and the reagents used are commercially available reagents unless otherwise specified. Example 1: Preparation of the combined vaccine YCWP+DLNP-HPV E7 1. Preparation of cationic lipid nanoparticles loaded with mRNA encoding HPV E7 (DLNP-HPV E7) DOTAP, DSPC, cholesterol, ALC-0159, and ALC-0315 were dissolved in anhydrous ethanol (DOTAP:DSPC molar ratio 11.5:34.8; DOTAP:cholesterol molar ratio 11.5:9.4; DOTAP:ALC-0159 molar ratio 11.5:42.7; DOTAP:ALC-0315 molar ratio 11.5:1.6), with a final volume of 1 mL. The mRNA encoding the HPV E7 antigen was dissolved in 50 mM citrate buffer at pH 4.0, resulting in an mRNA concentration of 167 μg / mL. The lipid ethanol phase and the mRNA aqueous phase were mixed at a volume ratio of 1:3 using a microfluidic device (R-SDM microfluidic chip, Myenna Corporation) at a total flow rate of 12 mL / min. The mixture was collected and rinsed with PBS (pH 10.5). 7.4) Dialyze at 4℃ for 6 hours, changing the dialysate every 2 hours. After dialysis, filter the solution through a 0.22μm filter membrane to obtain DLNP-HPV E7 with uniform particle size, and store at 4℃ for later use. 2. Preparation of YCWP Take commercially available brewing yeast ( Saccharomyces cerevisiaeActive dry yeast was activated with sterile water and washed twice. 10g of yeast was mixed with 100mL of 1M NaOH solution and heated in an 80℃ water bath for 1 hour with appropriate stirring. After heating, the mixture was centrifuged at 2000g for 10 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 100mL of pure water, and the pH was adjusted to 4.5 with HCl. After incubation in a 55℃ water bath for 1 hour, the mixture was centrifuged at 2000g for 10 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with pure water, three times with isopropanol, and three times with acetone. After each wash, the precipitate was collected by centrifugation at 2000g for 10 minutes. The washed precipitate was air-dried overnight in a fume hood to obtain yeast cell wall granules (YCWP).

[0020] The dried YCWP was resuspended in sterile PBS and vortexed to disperse it fully, preparing a stock solution of 10 mg / mL, which was then stored at 4°C for later use.

[0021] 3. Preparation of YCWP+DLNP-HPV E7 complex Mix 0.1 mL of 10 mg / mL yeast cell wall granule stock solution with 2 mL of DLNP-HPV E7 (the mass-to-volume ratio of YCWP to DLNP-HPV E7 is 1:2 mg:mL), incubate overnight at 4°C, centrifuge to remove the supernatant, and obtain the YCWP+DLNP-HPV E7 complex.

[0022] Example 2: Preparation of PEI-YCWP+LNP-HPV E7 combined vaccine 1. Preparation of PEI-modified yeast cell wall particles (PEI-YCWP) The YCWP (5 mg) prepared in step 2 of Example 1 was resuspended in 10 mL of water, 0.4 mL of 1 mg / mL potassium periodate solution was added, and the mixture was stirred in the dark at room temperature for at least 6 hours. The oxidized YCWP sample was washed three times with water and immediately used for reductive amination synthesis. Polyethyleneimine (PEI, 25000 Da) and 10 mL of water were added to the oxidized YCWP (1 μmol PEI / mg YCWP), resuspended, and mixed overnight at room temperature. The sample was washed three times with water, 0.1 g of sodium borohydride was added, and incubated at room temperature for 48 hours. The reduced sample was washed with water, resuspended overnight in 75% ethanol, sterilized, aseptically washed three times with physiological saline, and then resuspended with PBS to obtain a PEI-YCWP stock solution of 10 mg / mL 0.2 mL. 2. Preparation of LNP-HPVE7 ALC-0315, DSPC, cholesterol, and ALC-0159 were dissolved in anhydrous ethanol (molar ratio of ALC-0315 to DSPC: 46.3:9.4, ALC-0315 to cholesterol: 46.3:42.7, ALC-0315 to ALC-0159: 46.3:1.6), with a final volume of 1 mL. The mRNA encoding HPV E7 was dissolved in 50 mM citrate buffer (pH 4.0), resulting in an mRNA concentration of 167 μg / mL. The lipid ethanol phase and the mRNA aqueous phase were mixed at a volume ratio of 1:3 using a microfluidic device (R-SDM microfluidic chip, Myenna Corporation) at a total flow rate of 12 mL / min. The mixture was collected and concentrated using a 50 kDa ultrafiltration tube at 4°C. The solution was centrifuged at 3500 g for 30 minutes until the background ethanol concentration was ≤1%. The concentrate was then purified with PBS (pH 10.5). 7.4) Dilute to 4 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C for later use.

[0023] 3. Preparation of PEI-YCWP+LNP-HPV E7 complex Mix 0.1 mL of 10 mg / mL PEI-YCWP stock solution with 2 mL of LNP-HPV E7 (the mass-to-volume ratio of PEI-YCWP to LNP-HPV E7 is 1:2 mg:mL), incubate overnight at 4°C, centrifuge to remove the supernatant, and obtain the PEI-YCWP+LNP-mRNA complex, which is stored at 4°C for later use.

[0024] 4. Complex characterization (1) Particle size and potential measurement The YCWP, DLNP-HPV E7, and YCWP+DLNP-HPV E7 complex samples from Example 1, and the PEI-YCWP, LNP-HPV E7, and PEI-YCWP+LNP-HPV E7 complex samples from Example 2 were diluted to 100 μg / mL with sterile PBS and measured using a Zetasizer Nano ZS laser particle size analyzer (Malvern Panalytical). Measurement parameters were set as follows: temperature 25°C, equilibration time 12 seconds, detection angle 173°, with each sample repeated three times. Zeta potential (mV), particle size (nm / μm), and polydispersity index (PDI) were recorded.

[0025] The potential detection results are shown below. Figure 1 , 2 The figure shows that the zeta potential of YCWP is approximately -9.5 mV, while the potential of PEI-modified PEI-YCWP is +29 mV, confirming that PEI was successfully coated onto the YCWP surface. The zeta potential of LNP-HPV E7 is approximately -3 mV. Figure 2After co-incubation with PEI-YCWP, the E7 potential of the complex PEI-YCWP+LNP-HPV is approximately +4mV. Figure 1 Similarly, the zeta potential of DLNP-HPV E7 is +9mV, and the potential of the complex YCWP+DLNP-HPV E7 after co-incubation with YCWP (-9.5mV) is approximately +5.7mV, indicating that the adjuvant component and the antigen component successfully bind through electrostatic adsorption.

[0026] Particle size analysis showed that the hydrated particle size of LNP-HPV E7 was approximately 78.75 nm, with a polydispersity index (PDI) of 0.04, indicating that the antigen component had a uniform particle size distribution. The particle size of DLNP-HPV E7 was 105.2 nm, with a PDI of 0.14. The particle size of YCWP was 3383 nm, with a PDI of 0.15. The particle size of PEI-YCWP was 4241 nm, with a PDI of 0.2.

[0027] The scanning electron microscope results are shown in Figure 3 , 4 The figure shows that YCWP is a spherical micron-sized particle with a porous surface, while DLNP-HPV E7 is a nano-sized spherical particle that adheres to the surface of YCWP, forming a structurally stable YCWP+DLNP-HPV E7 complex. LNP-HPV E7 is a nano-sized spherical particle with a size of about 78 nm, which is extensively attached to the surface of PEI-YCWP, forming a structurally stable PEI-YCWP+LNP-HPV E7 complex. The above results confirm the physical binding of the adjuvant and the antigen.

[0028] (2) Transfection efficiency The complexes YCWP+DLNP-luciferase and PEI-YCWP+LNP-luciferase were prepared according to the methods in Examples 1-2, except that the HPV E7 gene was replaced with a luciferase reporter gene (UniProt, number PO8659). RAW 264.7 cells were seeded in six-well plates one day in advance at a concentration of 8 × 10⁻⁶ cells / well. 5 / well. On the second day, 3 μg of the corresponding reagent from the following groups was transferred to each well: PBS group, positive control lipo6000 + luciferase mRNA (lipo6000), mRNA-luciferase group, LNP-luciferase group, DLNP-luciferase group, YCWP group, YCWP + DLNP-luciferase group, and PEI-YCWP + LNP-luciferase group. After transfection, cells were cultured for 24 hours to allow for sufficient luciferase expression. After culture, the supernatant was aspirated, and cells were washed 1-2 times with pre-cooled PBS. An appropriate amount of reporter gene cell lysis buffer (RG005-1) was added to each well. After complete lysis, the lysis buffer was collected and centrifuged at 4°C to obtain the supernatant. 50 μL of the cell lysis supernatant was taken, and 100 μL of firefly luciferase detection reagent (RG005-2) equilibrated to room temperature was added. The relative light units (RLU) value was immediately read on a chemiluminescence analyzer to evaluate the luciferase expression level in each group. See results Figure 5 The figure shows that in RAW264.7 cells, the luciferase expression level in the PEI-YCWP+LNP-luciferase group was about twice that of the LNP-luciferase group, and the luciferase expression level in the YCWP+DLNP-luciferase group was also about twice that of the DLNP-mRNA group, suggesting that YCWP or PEI-YCWP enhances cellular uptake or endosome escape and improves translation efficiency.

[0029] Example 3: In vivo immune response experiment of combined vaccine 1. Establishment of an animal model of cervical cancer Female C57BL / 6 mice aged 6-8 weeks were subcutaneously inoculated with 2×10⁻⁶ mice on the right back. 5 TC-1 cells (a cell line established by co-transformation of primary lung epithelial cells from C57BL / 6 mice with HPV16E6, E7 oncogenes and activated H-ras gene, suspended in 100μL PBS); when the tumor grows to about 5mm in diameter (around day 12), the tumor-bearing mice are randomly divided into groups for the experiment.

[0030] 2. Mice were randomly divided into 7 groups (n=8 per group): PBS group, DLNP-HPV E7 group, LNP-HPV E7 group, PEI-YCWP group, YCWP group, YCWP+DLNP-HPV E7 group, and PEI-YCWP+LNP-HPV E7 group. The day of tumor inoculation was designated as day 0. Vaccination began on day 12, with intramuscular injections every 7 days for a total of 3 times. Each injection consisted of 100 μg of YCWP or PEI-YCWP, 100 μL of DLNP-HPV E7 or LNP-HPV E7, or 100 μL of YCWP+DLNP-HPV E7 or PEI-YCWP+LNP-HPV E7. Mice were sacrificed seven days after the last administration for relevant tests.

[0031] Tumor growth and curves as follows Figure 6 As shown in the figure, the tumors in the YCWP+DLNP-HPV E7 group and the PEI-YCWP+LNP-HPV E7 group were smaller than those in other experimental groups. This indicates that the compound vaccine of the present invention, which uses yeast cell wall particles as adjuvant, can effectively inhibit the tumor growth of TC-1 tumor-bearing mice. Among them, the PEI-modified platform (PEI-YCWP+LNP-HPV E7) has a more significant tumor-inhibiting effect. In the YCWP+DLNP-HPV E7 group, the tumors of some mice completely regressed (1 / 8 mice), and in the PEI-YCWP+LNP-HPV E7 group, the tumors of some mice completely regressed (3 / 8 mice). This shows that after the adjuvant and antigen mRNA are combined, the anti-tumor immune response can be synergistically activated, which can significantly inhibit the growth of HPV-positive tumors, and the PEI-modified compound vaccine has a better anti-tumor effect.

[0032] 3. Tumor tissue ELISPOT test Take a sterile 96-well ELISPOT plate, add anti-IFN-γ capture antibody (5 μg / mL, 100 μL / well), coat overnight at 4°C, wash 3 times with PBST, block with RPMI-1640 complete medium containing 10% FBS for 2 hours, mince the tumor tissue from each experimental group in step 2 (PBS group, DLNP-HPV E7 group, LNP-HPV E7 group, PEI-YCWP group, YCWP group, YCWP+DLNP-HPV E7 group, PEI-YCWP+LNP-HPV E7 group), digest with collagenase (1 mg / mL, 37°C, 30 min) to prepare a single-cell suspension, count the cells, and divide into 2×10⁶ cells per well. 5Cells were added to ELISPOT plates and stimulated with E7 peptide (final concentration 10 μg / mL). The plates were incubated at 37°C and 5% CO2 for 24 hours. After washing five times with PBST, biotin-labeled anti-IFN-γ detection antibody (2 μg / mL, 100 μL / well) was added, and the plates were incubated at 37°C for 2 hours. After washing five times with PBST, HRP-labeled streptavidin (100 μL / well) was added, and the plates were incubated at 37°C for 1 hour. After washing five times with PBST, AEC chromogenic buffer (100 μL / well) was added, and the plates were incubated at room temperature in the dark for 10-20 minutes. The reaction was terminated with deionized water after spots appeared. After drying at room temperature, the number of cells forming spots in each well was counted using an ELISPOT plate reader. Results were expressed as SFC / 10-1. 6 Cellular representation; See results Figure 7 Regarding local T cell responses in tumors, the number of IFN-γ ELISPOT spot-forming cells in the tumor tissue of the PEI-YCWP+LNP-HPV E7 group was 269 SFC / 10. 6 The number of cells increased by approximately 2.6 times compared to the PBS group (p<0.001), significantly higher than the LNP-HPV E7 alone group (p<0.001) and the PEI-YCWP alone group (p<0.0001); the number of cells in the YCWP+DLNP-HPV E7 group increased by approximately 1.5 times compared to the PBS group (p<0.001), indicating that the combined vaccine successfully broke the immunosuppression of the tumor microenvironment, allowing functional T cells to infiltrate into the tumor site and exert effector functions, and the PEI-YCWP+LNP-HPV E7 combined vaccine was more effective than the YCWP+DLNP-HPV E7 combined vaccine.

[0033] 3. Lymph node flow cytometry test TC-1 tumor-bearing mice that had undergone three immunizations in step 1 were sacrificed on day 7 after the last immunization. Inguinal and axillary lymph nodes were aseptically isolated. The lymph nodes were placed in RPMI-1640 medium containing 2% FBS, gently ground with the end of a syringe plunger, filtered through a 70μm cell sieve, and the cell suspension was collected. The cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS + 2% FBS. Cells were counted, and the cell density was adjusted to 1×10⁻⁶ cells / mL. 7 cells / mL.

[0034] Take 100 μL of cell suspension (1 × 10⁻⁶) 6 cells), add Fc blocking antibody (1μg / 10) 6 Cells), incubated at 4°C for 10 minutes. Add the following fluorescently labeled antibody mixture and incubate at 4°C in the dark for 30 minutes; after washing with PBS, use flow cytometry to detect and record the proportion of each cell population; Relevant immune markers include: acquired immunity-related marker CTL (IFN-γ+CD8+CD3+); innate immunity / trained immunity-related marker NK IFN-γ+ (IFN-γ+ in CD3- NK1.1+); and DC maturation marker CD86+ in CD11c+. See results Figure 8 Regarding the maturation of dendritic cells (DCs) in lymph nodes, the proportion of CD86+in CD11c+ in the PEI-YCWP+LNP-HPV E7 group was significantly higher than that in the PBS group (p<0.0001), and also significantly higher than that in the LNP-HPV E7 alone group and the PEI-YCWP alone group (p<0.0001). This indicates that DC maturation is highly dependent on the synergistic effect of YCWP adjuvant and LNP-mRNA antigen. Neither adjuvant alone nor antigen alone is sufficient to effectively induce DC maturation in lymph nodes, and the DC activation effect of the PEI-YCWP+LNP-HPV E7 combined vaccine is significantly better than that of the YCWP+DLNP-HPV E7 vaccine (p<0.0001).

[0035] Regarding the activation of CD8+ T cells in lymph nodes ( Figure 9 The proportion of IFN-γ+CD8+CD3+ cells in the PEI-YCWP+LNP-HPV E7 group was approximately 2.5 times higher than that in the PBS group (p<0.0001), and significantly higher than that in the LNP-HPV E7 alone group (p<0.05) and the PEI-YCWP alone group (p<0.01). The YCWP+DLNP-HPV E7 group was significantly higher than that in the DLNP-HPV E7 alone group (p<0.05) and the YCWP alone group (p<0.01). Among the combined vaccine groups, the PEI-YCWP+LNP-HPV E7 group was significantly higher than that in the YCWP+DLNP-HPV E7 group. These results indicate that the combined vaccine effectively initiates CD8+ T cell activation and induces its differentiation into IFN-γ secretory cells in lymph nodes, and the PEI-YCWP+LNP-HPV E7 combined vaccine is significantly superior to YCWP+DLNP-HPV E7 in inducing antigen-specific CTL responses.

[0036] Regarding the activation of NK cells in lymph nodes ( Figure 10The proportion of IFN-γ+in CD3-NK1.1+ cells in the PEI-YCWP+LNP-HPV E7 group reached the highest value among all groups, significantly higher than that in the PBS group (p<0.01). The proportion of IFN-γ+in CD3-NK1.1+ cells in the YCWP+DLNP-HPV E7 group was not significantly different from that in the PBS group, indicating that the PEI-YCWP+LNP-HPV E7 combined vaccine extended the activation range of NK cells from the spleen to the lymph nodes, suggesting that PEI-modified YCWP platform enhances the systemic activation capacity of NK cells, reflecting the functional gain of trained immunity at the level of innate immunity.

[0037] In summary, the PEI-YCWP+LNP-HPV E7 combined vaccine exerts its anti-tumor effect through a multi-level synergistic mechanism: at the dendritic cell (DC) level, CD86+in CD11c+ in lymph nodes was significantly elevated only in the PEI-YCWP+LNP-HPV E7 combined vaccine group, confirming that the synergistic effect of PEI-YCWP adjuvant and LNP-HPV-E7 antigen is a prerequisite for DC maturation; at the T cell level, the proportion of IFN-γ+CD8+CD3+ cells in lymph nodes and the number of IFN-γ ELISPOT spot-forming cells in tumor tissue were significantly increased in the combined vaccine group, confirming that the combined vaccine effectively initiated the activation, differentiation, and tumor infiltration of CD8+ T cells. PEI-YCWP+LNP-HPV-E7 can significantly enhance the IFN-γ response of NK cells in lymph nodes, achieving functional activation of NK cells in tumor-draining lymph nodes. As the core site of initial T cell activation, early activation of NK cells in lymph nodes is of great significance for establishing an anti-tumor immune microenvironment and promoting subsequent adaptive immune responses. The above results demonstrate for the first time in vivo that the mRNA-LNP combination vaccine with yeast cell wall particles as adjuvant can systematically activate DC maturation, CD8+ T cell response and NK cell function through the synergistic effect of YCWP adjuvant-induced training immunity and LNP-HPV E7 antigen-induced adaptive immunity, thereby achieving multi-level immune activation from innate immunity to adaptive immunity. Furthermore, PEI modification is a key optimization step to enhance the systemic immune activation capability of this combination vaccine platform.

[0038] Example 4: Evaluation of the effect of combined vaccine on bone marrow HSPC metabolic reprogramming 1. Bone marrow HSPC analysis On day 7 post-final immunization, femurs and tibias were harvested from mice in each group (PBS group, PEI-YCWP group, YCWP group, LNP-HPV E7 group, DLNP-HPV E7 group, YCWP+DLNP-HPV E7 group, PEI-YCWP+LNP-HPV E7 group). The bone marrow cavity was aseptically isolated, washed with PBS containing 2% FBS, and bone marrow cell suspension was collected. The suspension was filtered through a 70μm cell sieve to remove bone fragments, centrifuged at 400g for 5 minutes, and the supernatant was discarded. ACK erythrocyte lysis buffer was added, and the cells were incubated at room temperature for 3 minutes to lyse. Lysis was terminated by adding an equal volume of PBS, centrifuged at 400g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in PBS + 2% FBS, counted, and the cell density adjusted to 1×10⁶ cells / mL. 7 cells / mL.

[0039] Take 100 μL of cell suspension (1 × 10⁻⁶) 6 cells), add Fc blocking antibody (1μg / 10) 6 Cells were incubated at 4°C for 10 minutes. Then, the following fluorescently labeled antibody combination was added: Lineage, Sca-1, c-Kit, CD150, CD48, CD34, and CD135, and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0040] Obtain additional bone marrow cells and adjust the density to 1×10⁻⁶. 6 Cells / mL, add 2-NBDG to a final concentration of 50 μM, incubate at 37°C in the dark for 30 minutes; wash twice with PBS, then perform surface antibody staining as described above, and detect the mean fluorescence intensity (MFI) of 2-NBDG for each subpopulation by flow cytometry.

[0041] The gating strategies for each subgroup of HSPC are as follows: LSK (Lin - Sca-1 + c-Kit + MPP (CD150 in LSK) - CD48 + CD34 + CD135 + Record the proportion of each subpopulation in LSK cells and the MFI of 2-NBDG in MPP.

[0042] The results are as follows Figure 11 , 12As shown, compared with the PBS control group, the proportion of MPP increased in all groups except the PBS group, with the YCWP group showing the highest proportion. MPP 2-NBDG uptake was significantly enhanced only in the PEI-YCWP+LNP-HPV E7 group relative to the PBS group (p<0.0001); the other experimental groups were lower than the PBS group or showed no significant difference from the PBS group. These results indicate that the PEI-YCWP+LNP-HPV E7 combined vaccine can induce metabolic reprogramming of MPP at the bone marrow hematopoietic stem cell level, while PEI-YCWP or LNP-HPV E7 alone cannot achieve this effect.

[0043] Example 5: Study on the mechanism of synergistic activation of DC signaling pathway by the combined vaccine PEI-YCWP+LNP-HPV E7 1. Induced differentiation of BMDC Female C57BL / 6 mice aged 6-8 weeks were euthanized by cervical dislocation and then disinfected by immersion in 75% ethanol for 5 minutes. Under aseptic conditions, the bilateral femurs and tibias were separated, and the epiphyses were cut. The medullary cavity was repeatedly flushed with pre-cooled sterile PBS using a sterile syringe (1 mL, 26 G needle) until the bone ends turned white. The flushing fluid was collected and filtered through a 70 μm cell sieve to remove bone fragments and tissue blocks. The filtrate was centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The precipitate was washed once with 5 mL of sterile PBS, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Add 3 mL of ACK erythrocyte lysis buffer, incubate at room temperature for 3 minutes to lyse erythrocytes, then add 10 mL of RPMI-1640 medium containing 10% FBS to terminate lysis; centrifuge at 400 g for 5 minutes, discard the supernatant; resuspend the pellet in complete medium (RPMI-1640 containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL GM-CSF, 10 ng / mL IL-4), and adjust the cell density to 1 × 10⁻⁶ cells / mL. 6 Cells / mL. Inoculate 10 mL of the cell suspension into 10 cm bacterial culture dishes (non-TC treated) and incubate at 37°C in a 5% CO2 incubator. On day 3, add 5 mL of fresh complete culture medium containing GM-CSF and IL-4. On day 6, gently pipette and collect the semi-suspended cells, centrifuge at 400g for 5 minutes, resuspend in fresh complete culture medium, and adjust the density for later use. On day 7, under a microscope, numerous semi-suspended, star-shaped or dendritic BMDCs will be visible. Gently pipette and collect the cells, stain with trypan blue to count cell viability (should be >90%), and adjust the cell density to 2 × 10⁻⁶ cells / mL. 6 cells / mL available for later use.

[0044] 2. Stimulus program BMDCs induced to differentiate on day 7 were seeded into 6-well plates, 2 × 10⁶ per well. 6Cells were incubated overnight (12-16 hours) at 37°C in a 5% CO2 incubator with 2 mL of complete culture medium. The supernatant was discarded the next day, and the following treatment preparations were added to each group, with 3 replicates per group. The stimulation time was 15, 30, 60, and 120 minutes, and the cells were plated independently at each time point. G1: PBS group: Add an equal volume of sterile PBS (pH 7.4) G2: Empty LNP group: LNP (without mRNA, 20 μL / well, equivalent to the lipid mass of the LNP-mRNA group) was added. The preparation method of LNP is the same as step 2 of Example 2, except that mRNA is not added. G3: PEI-YCWP group: PEI-YCWP (final concentration 50 μg / mL) was added. G4: LNP-HPV E7 group: LNP-HPV E7 (containing 5 μg mRNA / mL) was added. G5: Combined vaccine group: PEI-YCWP+LNP-HPVE7 added (PEI-YCWP final concentration 50μg / mL, LNP-HPVE7 final concentration 5μg mRNA / mL). 3. Phosphorylation flow cytometry detection Upon reaching the designated time point, add 2 mL of preheated fixative directly to each well, mix gently, and fix at 37°C for 10-15 minutes. After fixation, transfer the liquid from the wells to 15 mL centrifuge tubes using a pipette. Rinse the bottom of each well once with 2 mL of PBS, combine the solutions in the same centrifuge tube, centrifuge at 400 g for 5 minutes, discard the supernatant, add 1 mL of pre-chilled True-Phos™ Perm Buffer (BioLegend), gently disperse the cells, and incubate on ice for 30 minutes. During incubation, gently vortex every 10 minutes. After fixation, permeabilize the cells with True-Phos™ Perm Buffer, stain with anti-CD11c-FITC, p-Syk-PE, and p-IRF3-APC antibodies, and detect phosphorylation levels by flow cytometry.

[0045] The results are as follows Figure 13 , 14 As shown, the fold change in p-IRF3 phosphorylation level in the G5:combined vaccine group at 30 minutes and 120 minutes was significantly higher than that in the PEI-YCWP group (p<0.05) and the LNP-HPV E7 group (p<0.01), indicating that the IRF3 / type I interferon pathway was synergistically activated and the signal duration was prolonged.

[0046] The phosphorylation levels of p-Syk were significantly increased in both the PEI-YCWP group and the combined vaccine group (p<0.001), with no significant difference between the two groups. The LNP-HPV E7 group showed no change, demonstrating that PEI-YCWP independently activates the Dectin-1 pathway.

[0047] The results indicate that the synergistic effect is pathway-selective, mainly concentrated on the IRF3 / Type I interferon axis, rather than global signal enhancement.

[0048] Example 6: In vitro validation of PEI-YCWP+LNP-HPV E7-induced training immunization 1. This experiment uses the classic "training-resting-challenge" trained immune model. BMDC was laid on a 24-hole plate (5×10). 5 Cells / well, 500 μL complete medium), were added to each group according to the following groups. Stimulation was performed at 37°C and 5% CO2 for 24 hours. After stimulation, the supernatant was discarded, and the cells were gently washed once with 500 μL pre-warmed PBS. 500 μL of fresh complete medium (without irritants) was added, and the cells were incubated at 37°C and 5% CO2 for 7 days, replacing half the medium (250 μL fresh medium) every 3 days. After incubation, the supernatant was discarded, and either 500 μL of fresh complete medium containing LPS (final concentration 100 ng / mL) or PBS (control group) was added. The cells were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the supernatant was collected, centrifuged at 800g for 5 minutes to remove cell debris, and stored at -80°C for ELISA (TNF-α, IL-6, IL-12p70). Cells were collected and stained with anti-CD11c, CD40, CD86, and MHC II antibodies, and incubated at 4°C in the dark for 30 minutes. Cells were then resuspended in cell staining buffer and analyzed by flow cytometry. G1: PBS group: Add an equal volume of sterile PBS (pH 7.4) G2: LNP-HPV E7 group: LNP-HPV E7 (containing HPV E7 mRNA, 20 μL / well) was added. G3: PEI-YCWP group: PEI-YCWP (final concentration 50 μg / mL) was added. G4: Combined vaccine group: PEI-YCWP + LNP-HPV E7 added (PEI-YCWP final concentration 50μg / mL, LNP-HPVE7 final concentration 5μg mRNA / mL). G5: β-glucan group (final concentration 45 μg / mL); ELISA results as follows Figure 15-17As shown, the PEI-YCWP+LNP-HPV E7 group exhibited significantly higher TNF-α and IL-6 secretion after the LPS challenge compared to the PBS training group (p<0.0001), increasing by approximately 2.5 times. The PEI-YCWP and LNP-HPV E7 training groups also showed increases, but to a lesser extent than the PEI-YCWP+LNP-HPV E7 group. This indicates that dendritic cells trained with the combined vaccine can generate a stronger inflammatory factor response upon secondary stimulation, demonstrating the broad-spectrum enhancement of trained immunity. Regarding IL-12p70, only the PEI-YCWP+LNP-HPV E7 group showed a significant increase relative to the PBS group (p<0.001), suggesting that adjuvant training or antigen training alone can only induce "superficial trained immunity" (only enhancing the secretion of inflammatory factors such as TNF-α and IL-6), while combined vaccine training can endow dendritic cells with "deep trained immunity" or "high-quality trained immunity," i.e., acquiring Th1 polarization capacity (IL-12p70 secretion). IL-12p70 is a key cytokine guiding CD4+ T cell differentiation towards Th1 and promoting CD8+ T cell function. Its enhanced secretion capacity after training suggests that dendritic cells (DCs) trained with the combined vaccine can more effectively guide antigen-specific T cells towards Th1 / CTL differentiation upon subsequent antigen encounters, rather than merely generating non-specific inflammatory responses. This result further confirms the synergistic effect of YCWP adjuvant and LNP-mRNA antigen components in training-induced immunity. The combined vaccine not only enhances the broad-spectrum innate immune response but also endows DCs with key capabilities for functional maturation, laying a functional foundation for subsequent efficient activation of adaptive immune responses.

[0049] Flow cytometry results as follows Figure 18-20As shown, the proportions of CD11c+CD40+ cells (p<0.0001), CD11c+CD86+ cells (p<0.01), and CD11c+MHC-II+ cells (p<0.0001) in the PEI-YCWP+LNP-HPV E7 group were significantly higher than those in the PBS training group. The proportions of CD11c+CD40+ cells (p<0.0001), CD11c+CD86+ cells (p<0.01), and CD11c+MHC-I+ cells (p<0.0001) were also significantly higher in the PEI-YCWP training group alone. The proportion of CD11c+MHC-II+ cells (p<0.001) was significantly higher in the LNP-HPV E7 training group alone (p<0.01). The above results demonstrate that the PEI-YCWP+LNP-HPV E7 combined vaccine in the classic LPS training immunization model significantly enhances the training immunization effect of BMDCs. After secondary LPS stimulation, the expression levels of key co-stimulatory molecules such as CD40, CD86, and MHC-II, as well as antigen-presenting molecules, in BMDCs trained with the combined vaccine were significantly upregulated, with the upregulation magnitude being significantly greater than that in the PEI-YCWP alone training group and the LNP-HPV E7 alone training group. This indicates a synergistic effect between the YCWP adjuvant and the LNP-mRNA antigen component in inducing training immunization. The combined vaccine endows dendritic cells with stronger antigen-presenting capacity and co-stimulatory function, laying a solid cellular foundation for the subsequent effective activation of adaptive immune responses. Furthermore, these results further confirm the unique advantages of yeast cell wall particles as an mRNA vaccine adjuvant in inducing training immunization.

[0050] 2. BMDC-trained immunity + LNP-mRNA challenge The experimental method was the same as in step 1 for training the classic immune model. During the challenge period, LPS was replaced with LNP-HPV E7 (final concentration 5 μg mRNA / mL). After stimulation for 24 hours, the supernatant and cells were collected. Grouped as follows: G1: PBS group: Add an equal volume of sterile PBS (pH 7.4) G2: Empty LNP group: Add LNP (without mRNA, 20 μL / well, equivalent to the lipid mass of the LNP-mRNA group. The preparation method of LNP is the same as step 2 of Example 2, except that mRNA is not added). G3: PEI-YCWP group: PEI-YCWP (final concentration 50 μg / mL) was added. G4: β-glucan group (final concentration 45 μg / mL); ELISA results are shown in Figures 21 and 22. Regarding IL-12 p70, the PEI-YCWP training group showed 67.6 pg / mL, 2.4 times that of the PBS training group (p<0.0001), while the LNP training group alone showed no significant increase. Regarding IL-6, the PEI-YCWP training group showed a significant increase compared to both the PBS and LNP training groups (p<0.0001). Flow cytometry results are shown below. Figure 23 and 24 As shown, the proportion of CD11c+CD40+ in the PEI-YCWP training group was 49.8%, compared to 22.5% in the PBS training group (p<0.0001) and 43.4% in the LNP training group (p<0.05). The proportion of CD11c+CD86+ was also significantly higher in the PEI-YCWP training group than in the PBS training group (p<0.0001) and the LNP training group (p<0.05). These results indicate that PEI-YCWP training improved the response of DCs to subsequent LNP-HPV E7 vaccines from "phenotypic activation" to "functional reprogramming."

[0051] The above results indicate that PEI-YCWP alone can significantly enhance the response of BMDCs to subsequent LNP-HPV E7 antigen stimulation. Specifically, after PEI-YCWP training, the expression of co-stimulatory molecules such as CD40 and CD86 in DCs was significantly upregulated, indicating that training endowed DCs with a stronger ability to activate the second T cell signal, which is "phenotypic activation." More importantly, DCs trained with PEI-YCWP were able to secrete a large amount of IL-12p70 during the LNP-HPV E7 challenge, while the LNP-only training group, although it could increase the expression of CD40 and CD86 to a certain extent (phenotypic activation), could not induce IL-12p70 secretion (functional maturation). This key difference reveals the essential difference between "phenotypic activation" and "functional reprogramming": LNP-only training can only induce non-specific upregulation of DC surface molecules, while PEI-YCWP training can endow DCs with true functional maturation, that is, the ability to actively guide the Th1 immune response when encountering antigens. IL-12p70, a key cytokine guiding CD4+ T cell differentiation towards Th1 and promoting CD8+ T cell function, exhibits enhanced secretion capacity after training. This suggests that DCs trained with PEI-YCWP will be able to more effectively initiate antigen-specific T cell differentiation towards Th1 / CTL upon subsequent antigen encounters. These results fully demonstrate that training immunization with PEI-YCWP as an adjuvant can elevate the DC response to mRNA vaccines from "phenotypic activation" to "functional reprogramming," that is, from simple upregulation of surface molecules to the acquisition of functional cytokine secretion capacity. This provides direct in vitro evidence for yeast cell wall granules as an adjuvant for mRNA vaccines to enhance adaptive immune responses through training immunization.

Claims

1. An mRNA conjugate vaccine using yeast cell wall particles as adjuvant, characterized in that: Including yeast cell wall particles and cationic lipid nanoparticles loaded with tumor antigen mRNA; Alternatively, this could include PEI-modified yeast cell wall particles or lipid nanoparticles loaded with mRNA encoding tumor antigens.

2. The mRNA conjugate vaccine using yeast cell wall particles as adjuvant according to claim 1, characterized in that: The mass-to-volume ratio (mg:mL) of yeast cell wall particles to cationic lipid nanoparticles loaded with tumor antigen mRNA was 1:1-2. The mass-to-volume ratio (mg:mL) of PEI-modified yeast cell wall particles to lipid nanoparticles loaded with mRNA encoding tumor antigens was 1:1-2.

3. The mRNA conjugate vaccine using yeast cell wall particles as adjuvant according to claim 1, characterized in that: The cationic lipid nanoparticles loaded with mRNA encoding tumor antigens were prepared by dissolving (2,3-dioleoxypropyl)trimethylammonium chloride, distearate phosphatidylcholine, cholesterol, methoxy polyethylene glycol bis(tetradecyl acetamide), and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) in anhydrous ethanol. The lipid mixture was then mixed with a citrate buffer solution encoding tumor antigen mRNA using a microfluidic device. The mixture was dialyzed and filtered through a 0.22 μm filter for sterilization.

4. The mRNA conjugate vaccine using yeast cell wall particles as adjuvant according to claim 3, characterized in that: The molar ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to distearylphosphatidylcholine is 10-12:30-35; the molar ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to cholesterol is 10-12:9-10; the molar ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to methoxy polyethylene glycol bis(tetradecyl acetamide) is 10-12:40-45; and the molar ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) is 10-12:1-2.

5. The mRNA conjugate vaccine using yeast cell wall particles as adjuvant according to claim 2, characterized in that: The molecular weight of PEI is 25,000 Da.

6. The use of the mRNA complex vaccine with yeast cell wall particles as adjuvant as described in any one of claims 1-5 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that: The tumor is cervical cancer.

8. The application according to claim 7, characterized in that: The mRNA nucleotide sequence encoding the tumor antigen is shown in SEQ ID NO:1.

Images